In many instances, it is desirable to determine the presence and the amount of a specific material in solution (the ‘medium’). Surface-based assays rely on the interaction of the material to be assayed (the ‘analyte’) with a surface that results in a detectable change in any measurable property. (For the purpose of this patent, we will define ‘analyte’ as the material to be assayed; this could be a small molecule, a large molecule such as a protein or DNA, a cell, or a collection of cells, an organism such as a bacterium or virus. ‘Analyte-specific receptor, or ‘recognition element’ will be used to refer to that complementary element that will bind its partner analyte; this could be a molecule or collection of molecules, a biomolecule or collection of biomolecules, such as a protein or DNA, a groove or templated depression on the substrate that has the complementary geometry and/or interaction). In general, in order to assay a specific analyte, the surface is modified so as to offer the appropriate chemical interaction. In immunoassays, for example, one takes advantage of the specificity of the antibody-antigen interaction: A surface can be coated with an antigen in order to assay for the presence of its corresponding antibody in the solution. Similarly, a strand of deoxyribonucleic acid (DNA) can be attached to a substrate and used to detect the presence of its complementary strand in solution. In any of these cases, the occurrence of binding of the analyte to its recognition element on the surface, which thus identifies the presence of the specific analyte in solution, is accompanied by a detectable change. For example, the binding can produce a change in the index of refraction at the interfacial layer; this can be detected by ellipsometry or surface plasmon resonance. Alternatively, the bound analyte molecules may emit light; this emission can be collected and detected, as is the case for fluorescence-based sensors. Non-optical signals may also be used, as in the case of radio immunoassays and acoustic wave sensing devices.
Diffraction is a phenomenon that occurs due to the wave nature of light. When light hits an edge or passes through a small aperture, it is scattered in different directions. But light waves can interfere to add (constructively) and subtract (destructively) from each other, so that if light hits a non-random pattern of obstacles, the subsequent constructive and destructive interference will result in a clear and distinct diffraction pattern. A specific example is that of a diffraction grating, which is of uniformly spaced lines, typically prepared by ruling straight, parallel grooves on a surface. Light incident on such a surface produces a pattern of evenly spaced spots of high light intensity. This is called Bragg scattering, and the distance between spots (or ‘Bragg scattering peaks’) is a unique function of the diffraction pattern and the wavelength of the light source. There is a unique correspondence between a specific pattern and the pattern of the light that is diffracted by it (the ‘diffraction image’), although in practice, diffraction is best illustrated by using periodic patterns, because these yield easily recognized diffraction images of clearly defined regions of high and low light intensity.
Diffraction techniques are commonly used in studies of molecular structure; specifically, X-ray diffraction is used in the identification of chemical compounds and in the determination of protein structures. However, the principle of diffraction, especially in the optical domain, has rarely been invoked for use in assays.
U.S. Pat. No. 4,647,544 (Immunoassay using optical interference detection) describes a light optical apparatus and method, in which a ligand, or an antibody, is arranged in a predetermined pattern, preferably stripes, on a substrate, and the binding between ligand and antiligand, or between an antibody and an antigen, is detected by an optical detector set at the Bragg scattering angle, which is expected to arise due to optical interference. The pattern of ligand or antibody is created by first laying out a uniform layer of antibody on a substrate, then deactivating sections of this coverage.
U.S. Pat. No. 4,876,208 (Diffraction immunoassay apparatus and method) describes the apparatus and reagents for an immunoassay based on a silicon or polysilicon substrate with a pattern of evenly spaced lines of a biological probe (a ‘biological diffraction grating’) to which binding can take place, The pattern is created by first coating the substrate with an even layer of antibodies, then deactivating regions by the use of a mask and of ultraviolet (UV) lights. This idea is extended to the assay of DNA in U.S. Pat. No. 5,089,387 (DNA probe diffraction assay and reagents), which describes a biological diffraction grating, and a process for its manufacture by first immobilizing a uniform layer of hybridizing agent on a smooth surface, and then exposing this surface to UV radiation through a mask with diffraction grating lines. The UV exposure deactivates the hybridizing agent, leaving a pattern of lines of active hybridizing agents.
In the apparatus described in these two patents, the presence and quantity of analyte is derived from the signal arriving at the detector positioned at one of the Bragg scattering angles. The detection scheme utilizes a photomultiplier tube or photodiode, miscellaneous optics and lock-in amplification that are well-known in the art of detecting small light signals.
U.S. Pat. No. 5,922,550 (Biosensing devices which produce diffraction images) describes a device and method for detecting and quantifying analytes in a medium based on having a predetermined pattern of self-assembling monolayer with receptors on a polymer film coated with metal. The size of the analytes are of the same order as the wavelength of transmitted light, and the diffraction pattern that arises due to binding is strong enough to be visible to the eye. This patent also describes a method of producing the patterned surface by microcontact printing of the self-assembled monolayer of receptors on a metal-coated polymer. This is extended to the case of a predetermined pattern of receptors (not necessarily self-assembling) in U.S. Pat. No. 6,060,256 (Optical Diffraction Biosensor). The technique of microcontact printing of self-assembled monolayers on a metal substrate is described in U.S. Pat. No. 5,512,131 (Formation of microstamped patterns on surfaces and derivative articles).
Microcontact printing is a technique of forming patterns of micrometer dimensions on a surface using an elastomeric stamp; the material to be patterned serves as the “ink” and is transferred by contacting the stamp to the surface. Microcontact printing of proteins on silicon, silicon dioxide, polystyrene, glass and silanized glass is reported in Bernard, A; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H.; “Printing Patterns Of Proteins” Langmuir (1998), 14, 2225-2229.
To utilize diffraction techniques in surface-based assays, it is important to be able to produce a material patterned with receptors, and the five patents discussed above have outlined their ways of doing so. In addition, other techniques that exist in the literature may be adaptable for patterning. For example, using photolithographic techniques, oligonucleotides have been immobilized on a substrate in arrays such that each array is a distinct species. U.S. Pat. Nos. 5,831,070 and 5,599,695 show how this is done through the use of deprotection agents in the gas phase. This approach has not been used in the creation of patterns for diffraction assays, but can be adapted for such with the design of an appropriate mask.
To summarize the prior art, it has been shown that diffraction of light that occurs upon binding of an analyte to its partner receptor that has been patterned on a substrate can be used for an assay. In U.S. Pat. Nos. 4,647,544 and 4,876,208, the pattern of evenly spaced lines (grating) is used, and the signal is detected by a detector at the expected location of the Bragg peaks. The diffraction intensity is low enough and necessitates the use of a lock-in amplifier and associated optics and electronics for its detection. In U.S. Pat. Nos. 5,922,550 and 6,060,256, the analyte being assayed is large enough, so that the diffraction pattern is visible to the eye. The pattern on the surface consists of simple geometric elements such as circles of uniform size, and which are evenly spaced.
These patents which make up the prior art do not consider the importance of the type of pattern on the surface, upon which this invention is based. The present invention specifically refers to assays using the principles of diffraction wherein the surface pattern is different from the prior art of evenly spaced lines (a grating), or evenly spaced circles. The choice of design for the surface pattern can enhance the effectiveness of the assay, for example, by enhancing the signal.